1.1 Field of the Invention
The present invention relates generally to the fields of molecular biology. Disclosed are methods and compositions comprising DNA segments, and proteins derived from sea anemone species. More particularly, it concerns novel ShK toxin, toxin analogs, modified toxin analogs, and genes encoding the ShK toxin from Stichodactyla helianthus. Various methods for making and using these DNA segments, DNA segments encoding synthetically-modified ShK toxins, and native and synthetic ShK peptides are disclosed, such as, for example, the use of DNA segments as diagnostic probes; and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications.
1.2 Description of Related Art
A multitude of potassium (K) channels have been discovered in the past decade. The synergistic interplay of molecular biological and electrophysiological approaches has permitted the isolation, individual expression, and functional analysis of many K channels within the past five years. K channels can be conveniently though arbitrarily divided into voltage-gated and chemically-gated types. The voltage-gated channels primarily consist of delayed-rectifiers (DR) and inward-rectifiers. These are primarily important for modulating excitability and determining the rate of repolarization during action potentials. Chemically-gated K channels include ATP-inhibited, Ca-activated, and neurotransmitter-gated channels. These function in the long-term modulation of cell membrane potential, thereby affecting smooth muscle tone, synaptic excitability, neurotransmitter release, and other processes. While many types of K channel are widely distributed in various tissues of the body, one of the delayed-rectifier channels, Kv1.3, is almost exclusively located in T lymphocytes (Cahalan et al., 1991; Lewis and Cahalan, 1995). This lymphocyte K channel has been shown to be homo-oligomeric, in contrast with many DR channels in the nervous and muscular systems, which can exist as hetero-oligomers containing more than one subunit. For instance, in the rat brain, most DR channels are of the Kv1.2 and Kv1.1 types, and these two types of subunits also may be present in the same channel (Scott et al., 1994).
The mechanisms by which Kv1.3 channels affect lymphocyte proliferation are being investigated in several laboratories. The major evidence that they are involved is the ability of ChTx and margatoxin to inhibit lymphocyte proliferation and interleukin 2 production (Chandy et al., 1984; Price et al, 1989; Garcia-Calvo et al., 1993). Inhibition of Kv1.3 depolarizes the cell membrane sufficiently to decrease calcium influx, and this prevents elevation of free intracellular calcium concentration which is the stimulus for these two responses. Other K channel inhibitors such as TEA, 4-aminopyridine, and quinidine also inhibit proliferation, but they often have other effects as well. Other K channels like K(Ca) channels are also present, but their blockade does not inhibit interleukin release or proliferation (Leonard et al., 1992; 1995). The restricted tissue distribution of Kv1.3 and its immunosuppressive action upon T-cells has prompted several pharmaceutical companies to attempt development of specific Kv1.3 blockers for therapeutic use as immunosuppressants. Blockade of other DR K channels in the body is thought to be deleterious to health, with two possible exceptions. The Kv1.5 channel occurring in the myocardium, when blocked, leads to prolongation of the cardiac action potential. So there is interest in developing Kv1.5 channel-selective blockers as anti-arrhythmic agents. Second, selective inhibition of certain DR K channels in the hippocampus might be useful in enhancing memory in Alzheimer's patients (Lavretsky et al., 1992).
Until recently, K channel investigations were hampered by a paucity of selective neurotoxin probes, which have been so important for investigating sodium and calcium channels. But this has dramatically changed in the past few years. The sea anemone K channel toxins are the most recent addition to the K channel armamentarium. The dendrotoxins are relatively large peptides, and this has limited their utility in determining where they bind to the DR K channels, because of difficulties in preparing analogs or mutants of this toxin, of which the solid-phase synthesis has been recently accomplished. By exchanging functional domains of two DR channels, only one of which is sensitive to dendrotoxin, Stocker et al. (1991) showed that the external loop between S5 and S6 contains at least part of the dendrotoxin receptor. Using K channel mutants, Hurst et al. (1991) reached a similar conclusion. This region contains over 40 residues of sequence, and about half of these contribute towards pore formation. Besides the dendrotoxins, other known K channel toxins include the mast cell degranulating peptide (MCDP), various homologous scorpion peptides (Garcia et al., 1991), two sea anemone toxins (ShK and BgK toxins, Castaneda et al., 1995), and several as yet unpurified molluscan peptides.
Chandy et al. (1995) have shown that the scorpion K channel toxins (charybdotoxin as prototype) are potent blockers of Kv1.1, Kv1.2, and 1.3 Shaker type DR channels. While charybdotoxin (ChTx) also blocks maxi-type K(Ca) channels, some newer ChTx homologs including margatoxin lack K(Ca) channel blocking activity. The scorpion K channel toxins are valuable tools for investigating these ER channels as well as the maxi-K(Ca) channels. Since they are also rather rigid molecules, they are also proving useful as "molecular calipers" for measuring distances between K channel amino acid residues in the outer vestibule of these channels (Stocker and Miller, 1994; Chandy, 1995). A functional map of the interactive surface of the scorpion K channel blocker charybdotoxin has been derived by cloning, expressing, and testing numerous monosubstituted ChTx analogs (Park and Miller, 1992; Stampe et al., 1994). Eight residues (Ser10, Trp14, Arg25, Lys27, Met29, Asn30 and Tyr36) were identified as crucial for ChTx's channel-blocking function. Replacement of any of these residues increased the dissociation rate constant at least 8-fold. Thus, ChTx utilizes a combination of hydrophobic, H-bonding and ionic interactions in its interactions with the Shaker DR (Goldstein et al., 1994) and skeletal muscle maxi-K(Ca) channels (Siampe et al., 1994). The K-channel receptor site may be thought of as reciprocally endowed with the appropriate chemical features to accommodate these interactions.
Both of the two antiparallel B-sheets in the scorpion DR blocking toxins are within the C-terminal region. By preparing and testing various chimeric toxins of the homologous pharmacologically different scorpion toxins ChTx and iberiotoxin, it was shown that the C-terminal third of ChTx confers DR blocking activity, which suggests that residues in the C-terminal .beta.-sheet are interacting with the K channel (Giangiacomo et al., 1993). ShK toxin is structurally quite different from the scorpion toxins and our initial pharmacological data (next section) indicate that it also uses its helical portion to interact with the Shaker-type K channels.
Voltage-gated potassium (K.sup.+) channels regulate diverse biological processes (Chandy and Gutman, 1995). A short stretch of amino acids, the P-region, located Between the fifth and sixth transmembrane segments, contributes to the formation of the channel pore. Delineation of the spatial organization of the residues in the P-region would help define the structure of the ion conduction pathway and be valuable for understanding the mechanisms of ion permeation. Scorpion (ChTx, KTx) and sea anemone toxins (ShK) apparently interact strongly with residues in the P-region. It should be possible to deduce the spatial arrangement of the residues in the P-region by using these toxins as structural templates, provided the three-dimensional structures of the toxins is known. In preliminary studies using NMR and molecular modeling, The inventors have shown that four scorpion toxin-blockers of K.sup.+ channels, kaliotoxin (KTX), margatoxin (MgTX), noxiustoxin (NTX) and charybdotoxin (ChTX) have a similar tertiary fold.
Many times different molecules utilize the same functional groups to bind with their receptors. In the case of the Na-channel, the toxins tetrodotoxin and saxitoxin are heterocyclic organic compounds which utilize essential guanidinium functionalities to block Na channel function by binding to the Site I receptor (Catterall, 1980). Mu-conotoxins, short peptide toxins isolated from Conus venoms, also competitively bind to the same site I receptor. Interestingly, these toxins are able to discriminate between the tetrodotoxin/saxitoxin receptor on muscle and nerve sodium channels (Ohizumi et al., 1986). Structurally, these peptide toxins are highly constrained by three disulfide bonds which are utilized to correctly position a guanidinium functionality present on an invariant Arg residue (Arg13 in .mu.-CgTX GIIIA) for channel-blocking activity (Sato et al., 1991). Thus, in tetrodotoxin and saxitoxin, the essential binding features of .mu.-conotoxin have been naturally incorporated into a small organic type of scaffold. Design of similarly peptidomimetic compounds (but inhibiting Kv1.3 channels) is one major goal of the inventors' project.
Peptides are characteristically highly flexible molecules whose structure is strongly influenced by their environment (Marshall et al., 1978). Nature introduces conformational constraints such as disulfide bonds to help lock a molecule into the biologically active structure. These types of constraints and other structures such as .alpha.-helix, .beta.-sheet and reverse turns combine to form the architecture for a peptide/protein's three dimensional structure. The surface localization of turns in proteins, and the predominance of residues containing potentially pharmacophoric information has lead to the hypothesis that turns play a critical role in recognition events (Rose et al., 1985). The stability of .alpha.-helical conformations in peptides has also been found to be essential for biological activity in many different systems (Kaiser and Kezdy, 1983).
While the receptor binding domain of an antigenic site, toxin, or hormone may be relatively large, only a relatively small subpopulation of contact residues contribute most of the free energy decrease upon binding. For instance, co-crystallization of human growth hormone with the extracellular domain of its receptor using an Ala scan type of substitution approach has been utilized to identify critical residues providing large contributions to the binding energy of this interaction (Clackson and Wells, 1995). The receptor surface and protein ligand surface each contributed approximately 30 amino acid sidechain contact points. Using Ala-based substitutions at each of these contact points on the receptor, these researchers were able to determine that over 75% of the binding free energy was accounted for by two tryptophan residues. These functionally important residues on human growth hormone receptor make direct contact with those on human growth hormone.
This and other studies provide considerable optimism that it is possible to design small molecules incorporating at least some of the critical chemical groups crucial for interaction with a target receptor. These peptidomimetic compounds should have better use as drugs than the peptides or proteins which they resemble, because they will be more readily absorbed when administered orally, display little or no antigenicity, and be less susceptible to proteolytic attack.